Within the last decade it has become apparent that advantages are available from utilizing the specific interaction of the antibodies produced by the immune system or fragments thereof to confer a homing capability on a substance to be delivered to a particular target location in a subject. A common application is the use of immunotoxins which are conjugates between toxic materials, such as ricin or abrin, with an antibody preparation that is specific for a tissue against which the toxin is expected to react. Other examples include the conjugation of organic labels, such as fluorescent labels, to immunoglobulins in order to identify the location of certain target tissues.
Analogous conjugates have also been formed to confer immunogenicity on materials carrying epitopes to which antibodies are to be raised, but which lack sufficient size to be immunogenic. For example, a substantial amount of research has been done on the preparation of peptide vaccines by ligating short amino acid sequences to carrier proteins. This permits the relevant epitope to be used in quantity and to be synthesized chemically.
In each of these instances, it is necessary to find an efficient and nondestructive way to couple a protein subject to denaturation, i.e., the homing agent or carrier protein, with an "active" substance of interest. Conditions must be found which provide satisfactory amounts of product while maintaining the conformation of the protein.
Two basic approaches have been taken. The first uses linkers which become part of the conjugate. These linkers are homobifunctional or heterobifunctional, and include those capable of forming, for example, disulfide linkages through the thiol groups of cysteine moieties in the substrate proteins, or of the formation of amide linkages between N-terminal amino group or the amino side chains or lysine residues and activated acyl moieties such as succinimidyl esters. In general, this approach involves highly reactive functional groups on the linker and is reasonably facile with respect to the substrates for conjugation. However it is often useful to employ functional groups which may be less reactive, such as those capable of hydrazone formation.
A second approach, particularly useful in conjugating two protein moieties, uses a dehydrating agent such as a carbodiimide to effect the formation of, for example, new peptide bonds by reaction of a carboxyl moiety on one member of the conjugate with a free amino group on the other. In this case, the reagent does not become part of the conjugate. This reaction is not particularly facile since the carboxyl group is not activated; the carbodiimide provides the active intermediate and shifts the equilibrium by removing the elements of water to form the peptide bond.
Both approaches to conjugation have generally been conducted in aqueous solvents because the protein material forming the conjugate is easily denatured. Proteins are designed to be stable in an aqueous environment and are known to denature even in solvents, such as ethanol, which would be thought to be reasonably analogous to an aqueous medium. Also, protein conjugate components tend to be relatively insoluble in nonaqueous solvents.
Effecting a conjugation between proteins using functionalities which result in the elimination of water, i.e., dehydration, is therefore often done in aqueous medium. While workable, this clearly is not particularly facile nor efficient. Since relatively long reaction times are required, the opportunity for side reactions is also great.
It has previously been disclosed that polar aprotic solvents may be present in the reaction mixture without resulting in sufficient denaturation of product to undermine its utility. For example, in the conjugation procedure described in Mew. D., et al. J. Immunol (1983) 130: 1473-1477, hematoporphyrin is conjugated to antibody protein after an initial reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl (EDCI) using a total volume of approximately 2.5 ml of which 0.8 ml is dimethyl formamide (DMF). However, applicants are aware of no instance in which the conjugation has been successfully conducted in what amounts to a completely nonaqueous environment. The present invention demonstrates that such a reaction medium is not only nondenaturing during the course of the reaction, but is also advantageous in increasing the rate of reaction and the efficiency of coupling.